This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding acyl-CoA oxidase homologues in plants and seeds.
The first step in the beta-oxidation pathway is catalyzed by acyl-CoA oxidase, an oxidoreductase which requires FAD as a cofactor. Acyl-CoA oxidase acts on the CHxe2x80x94CH group of donors using oxygen as an acceptor molecule. It catalyzes the following reaction:
Long chain (8 and more) acyl-CoA+O2=trans-2,3-dehydroacyl-CoA+H2O2
Acyl-CoA oxidase (EC 1.3.3.6) has been widely studied in human tissues where it has been found localized in the peroxisomes. Acyl-CoA oxidase genes have also been described in other mammals, as well as in bacteria and yeast. The gene encoding a putative acyl-CoA oxidase from Hordeum vulgare has been isolated and expressed in vitro. Genomic sequencing projects have found homologous genes in Arabidopsis thaliana, Phalaenopsis sp. and Oryza sativa. Studies on the rat acyl-CoA oxidase gene indicate that two species of mRNA are produced by possible alternative splicing; Southern blots indicate a single copy gene (Osumi T., et al. (1987) Biol Chem 262:8138-8143). The two forms ofthe enzyme (acyl-CoA oxidase 1 and 2) differ only within a small region of about 30 amino acids, and contain the same number of amino acids. In the yeast Candida tropicalis, isozymes of acyl-CoA have been described (Picataggio, S. et al. (1991) Mol. Cell. Biol. 11:4333-4339). Gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. The two isozymes are differentially regulated and display unique substrate profiles and kinetic properties.
The instant cDNAs for acyl-CoA oxidase also show homology to an Arabidopsis thaliana chromosome 4 homologue of the TINY region of the Arabidopsis apetela2 gene (AP2) from amino acids 234-885 of the 895 amino acid sequence. Apetela2 is a homeotic gene involved in setting up or responding to concentric or overlapping fields within the flower primordium (Bowman, J. L. et al. (1989) Plant Cell 1:37-52). An apetela2 mutation (ap2-1) has a pleitotropic effect on seeds: Arabidopsis thaliana ap2-1 seed shape will vary from the normal oblong shape to a variety of aberrant shapes (Leon-Kloosterziel, K. M. et al. (1994) Plant Cell 6:385-392). The AP2 domain has been linked to increased seed size and protein content in seeds (Okamuro, J. et al., (1994) Proc. Natl. Acad. Sci. USA 94:7076-7081).
Accordingly, acyl-CoA oxidase is a key step in beta-oxidation of fatty acids in plants. Manipulation of this step in the breakdown of fatty acids may allow transgenic plants to accumulate significant amounts of unusual fatty acids in their seed. This would facilitate the development of transgenic plants that produce novel oils.
The instant invention relates to isolated nucleic acid fragments encoding acyl-CoA oxidase homologues. Specifically, this invention concerns an isolated nucleic acid fragment encoding an acyl-CoA oxidase homologue and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding an acyl-CoA oxidase homologue. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding acyl-CoA oxidase homologue.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an acyl-CoA oxidase homologue.
In another embodiment, the instant invention relates to a chimeric gene encoding an acyl-CoA oxidase homologue, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an acyl-CoA oxidase homologue, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an acyl-CoA oxidase homologue, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of an acyl-CoA oxidase homologue in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an acyl-CoA oxidase homologue; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of acyl-CoA oxidase homologue in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an acyl-CoA oxidase homologue.